Methods and kits for identifying scavengers of reactive oxygen species (ROS)

ABSTRACT

This invention relates to methods and kits for determining the level of H 2 O 2  inside a cell, and for determining whether a test compound has ability to scavenge a reactive oxygen species (ROS). The methods and diagnostic kits of this invention employ a cell containing a promoter which is inducible by an ROS, such as the H 2 O 2  -inducible KatA promoter of  Agrobacterium tumefaciens . The methods of this invention may also be used to select for new or improved ROS scavengers by expressing a library of test scavengers in cells which express a reporter from a ROS-inducible promoter and selecting for those cells whose level of ROS-inducible expression of the reporter is reduced.

REFERENCE TO RELATED APPLICATIONS

This is the U.S. National Stage of International Application No.PCT/SG02/00018, filed Feb. 5, 2002 (published in English under PCTArticle 21(2)), which in turn claims the benefit of U.S. ProvisionalPatent Application No. 60/266,657, filed Feb. 5, 2001. Both applicationsare incorporated herein in their entirety.

FIELD OF INVENTION

This invention relates to methods of identifying, and/or determining theROS-scavenging ability of, a compound with ROS-scavenging function.

BACKGROUND OF THE INVENTION

Aerobic metabolism in living organisms can lead to generation ofreactive oxygen species (ROS), which include hydroxyl radicals,superoxide anion, hydrogen peroxide and nitric oxide. Production of ROScan be due to various enzymatic and non-enzymatic processes. In aerobicorganisms, ROS are formed from the partial reduction of molecular oxygento water during oxidative metabolism. Bacterial cells produce endogenoushydrogen peroxide from the dismutation of superoxide or hydroxyl radicalas a product of the respiratory chain when oxygen is used as theterminal electron acceptor. Enteric bacteria (e.g. Salmonellatyphimurium and E. coli) encounter toxic levels of hydrogen peroxideproduced by macrophages during engulfment.

Under normal conditions, ROS may play an important role in differentbiological processes. However, when ROS are excessively produced undercertain unusual conditions, they can cause oxidative damage to DNA,proteins and lipids.

ROS have been implicated in the pathogenesis of many different diseasesituations as well as harmful conditions. These include aging, AIDS,atherosclerosis, cancer, cataracts, congestive heart failure, diabetes,inflammatory disorders, rheumatoid arthritis, and neuro-degenerativediseases such as Alzheimer's, Parkinson's, multiple sclerosis, andDown's syndrome, in addition to exposure to pollutants and ionizingirradiation.

Living organisms have developed different ways of coping with the ROS.The capacity of enzymatic or non-enzymatic antioxidants to quench theROS can help cells to defend against the oxidative stress. Therefore,antioxidants have been linked to and used for disease prevention.

Antioxidants may be proteins, such as ferritin, lactoferritin andtransferritin, or enzymes, such as superoxide dismutase, catalase andglutathione peroxidase. Nonenzymatic antioxidants may be macromolecules,such as albumin, copper-binding ceruloplasmin and hemoglobin, or smallmolecules which may be water-soluble antioxidants, such as vitamin C,uric acid and bilirubin or lipid-soluble antioxidants, such as vitaminE, carotenoids, retinoids and ubiquinol-10.

However, these natural defenses can be overwhelmed in many pathologicalstates. More potent antioxidants should be supplemented to deal with theoxidative stresses. Screening and assay methods are needed to identifypotent antioxidants; but the current methods are both time-consuming andexpensive. In addition, they cannot measure the intracellularantioxidant activities, which are more relevant to biologicalapplications. Therefore, a simple method that can measure theintracellular antioxidant activities and hence can be used to search forbetter oxidant scavenging molecules is of great importance in thepharmaceutical and nutraceutical fields.

The public has shown an increasing interest in the natural antioxidantscontained in dietary supplements, as antioxidants can give healthbenefits by preventing oxidative damage caused by ROS. Standardizedassays to assess antioxidant activities and distinguish differentantioxidants are useful. Such assay methods are useful to properlyassess and label antioxidant products. Such assays are also useful formeasuring activities of antioxidants for use as food supplements,natural products and drugs.

Farr (U.S. Pat. Nos. 5,585,252, 5,811,231 and 5,589,337) have describeduse of stress promoters fused to reporter genes to determine toxicity.

Catalase is a protein antioxidant. Catalases catalyze the dismutation ofhydrogen peroxide to water and oxygen. The primary role for catalases isto protect the cells against the damage caused by reactive oxygenspecies to cellular components, including nucleic acids, proteins andcell membranes. Catalases have been implicated to be important for thesurvival of some pathogenic bacteria during infection and even for thelife span of a multi-cellular organism.

Agrobacterium tumefaciens is a soil-borne plant pathogen that causescrown gall tumors on many plant species. A chromosomal gene katAencoding a catalase has been identified that is involved indetoxification of H₂O₂ released during Agrobacterium-plant interaction(Xu and Pan, 2000).

SUMMARY OF THE INVENTION

The present invention relates to a method for determining the ability ofa compound to remove an ROS. The method generally comprises: a)providing a cell containing an ROS-inducible promoter (RIP) which drivesexpression of a reporter gene. The reporter gene is heterologous to thepromoter to which it is operably linked. b) exposing the cell to acompound potentially able to remove the ROS. c) measuring a change inthe ROS-inducible expression level of the reporter gene in the cell whenthe cell is exposed to the compound. A reduction in the reporter proteinlevel would indicate that the compound is able to remove the ROS.

The invention further relates to a method for selecting a nucleic acidwhich encodes a protein-potentially able to remove an ROS. The methodgenerally comprises: a) Providing cells containing an ROS-inducedpromoter which drives expression of a reporter gene. The reporter geneis heterologous to the promoter to which it is operably linked. b)Introducing into the cells expression vectors containing differentnucleic acids, such as those found in a cDNA library, or in a librarywhere the nucleic acids have been mutagenized. These nucleic acidsencode proteins which are potentially able to remove the ROS. c)Measuring a change in the ROS-inducible expression of the reporter genein the cells when the nucleic acids are expressed. d) Selecting forcells with reduced ROS-inducible expression of the reporter gene. e)Isolating the nucleic acid from the cells with reduced ROS-inducibleexpression of the reporter gene. The nucleic acid isolated by such aprocedure likely encodes a protein able to remove the ROS.

In one embodiment, the different nucleic acids all encode proteins ableto remove ROS. By selecting for cells with the greatest degree ofreduction in the level of reporter protein, the most efficient ROSremover may be identified.

The method of the invention does not require that the cell be exposed toan external source of ROS. Rather, the ROS which induces theROS-inducible promoter may be intracellular. In one embodiment, theintracellular level of the ROS may be elevated by methods known in theart. The intracellular level of ROS such as H₂O₂ may also be induced byacid pH, especially in bacteria such as A. tumefaciens. In anotherembodiment, the intracellular level of the ROS may be madeconstitutively elevated by using a cell which has been geneticallymodified.

Cells containing such genetic modifications are known in the art and mayhave, for example, modified genes of the respiratory chain so that theredox balance of the cell is disturbed. Other genetic modifications mayinvolve knocking out functional enzymes which break down or remove ROSintracellularly, such as catalase, superoxide dismutase, alkylhydroperoxidase, and glutathione reductase.

As is clear from above, the method of the invention also does notrequire that the potential ROS-removing compound be exposed to the cellextracellularly. Rather, the compound, in this case a gene product, maybe expressed from a nucleic acid inside the cell.

In a preferred embodiment, the cell expressing the RIP-reporter does notexpress the functional native gene product, i.e. that which is naturallyexpressed from the promoter to which the reporter gene is operablylinked. Absence of the native gene function ensures that no complicatingmechanism such as a feedback loop interferes with the correlationbetween the ability of a compound to remove an ROS and the RIP-reporterexpression level.

In one embodiment, the ROS-inducible promoter is from a gene selectedfrom the group consisting of: AhpCF, Bcp, Dps, gor, KatA, KatB/AnkB,KatG, TrxB, human MAP kinase phosphatase 1 (MKP-1) genes; mammalianhic-5 genes, the isc operon; Escherichia coli zwf, fpr, fumC, micF, nfo,and sodA genes; Azotobacter vinelandii spr gene; Xanthomonas oryzae pvoryzae katX gene; rat and human haem oxygenase-1 (HO-1); yeast2-deoxyglucose-6-phophate phosphatase (DOG2); catalase; human manganesesuperoxide dismutase (MnSOD); rat glutathione S-transferase (GST); humaninterstitial collagenase (MMP-1); human glutathione peroxidase (GPX2);fish metallothionein (MT); and rat multidrug resistance type 1 (mdr1).

In another embodiment, the ROS is H₂O₂. Where it is desirable toidentify or select for H₂O₂-removing compounds, an H₂O₂-induciblepromoter is used. Such a promoter may be from the following genes:AhpCF, Bcp, Dps, gor, KatA, KatB/AnkB, KatG, TrxB, human MAP kinasephosphatase 1 (MKP-1) genes; mammalian hic-5 genes, and the isc operon.

In another embodiment, the cell used in the methods described above is abacterial cell. It is understood that if a bacterial cell is used, theROS-responsive promoter must function as such in bacteria and thereporter gene must encode a protein functional in bacteria. Likewise, ifa plant cell, a yeast cell, or a mammalian cell is used, theROS-responsive promoter and the reporter protein must be functional inthe particular chosen cell type.

The present invention also relates to diagnostic kits for determiningthe ability of a gene product to remove an ROS. Such a kit generallycomprises: a) a cell which contains an ROS-inducible promoter drivingexpressing of a reporter gene. The reporter gene is understood to beheterologous to the promoter to which it is operably linked. b) meansfor introducing in the cell a nucleic acid encoding a gene product; and(c) instructions for determining a reduction in ROS-inducible expressionof the reporter gene in the cell once the nucleic acid is expressed.This would indicate whether the gene product is able to remove the ROS.

The kit of the invention provides components for carrying out themethods of the invention. Accordingly, in one embodiment, the kitfurther contains means for measuring the level of the product of thereporter gene. The kit may further comprise means for elevating theintracellular level of the ROS in the cell. The cell provided in the kitmay be genetically modified to contain an elevated intracellular levelof an ROS, or may lack at least one naturally occurring ROS-removingactivity. The cell of the kit may also lack a gene encoding an activeenzyme such as catalase, superoxide dismutase, alkyl hydroperoxidase,and glutathione reductase.

The ROS-inducible promoter contained in the kit may be from a gene suchas AhpCF, Bcp, Dps, gor, KatA, KatB/AnkB, KatG, TrxB, human MAP kinasephosphatase 1 (MKP-1) genes; mammalian hic-5 genes, the bacterial iscoperon; Escherichia coli zwf, fpr, fumC, micF, nfo, soi28, and sodAgenes; Azotobacter vinelandii spr gene; Xanthomonas oryzae pv oryzaekatX gene; rat and human haem oxygenase-1 (HO-1); yeast2-deoxyglucose-6-phophate phosphatase (DOG2); catalase; human manganesesuperoxide dismutase (MnSOD); rat glutathione S-transferase (GST); humaninterstitial collagenase (MMP-1); human glutathione peroxidase (GPX2);fish metallothionein (MT); and rat multidrug resistance type 1 (mdr1).

In one embodiment, the kit is used to determine the ability of a certaincompound to remove H₂O₂. Preferably, the kit provides an H₂O₂-induciblepromoter from a gene such as AhpCF, Bcp, Dps, gor, KatA, KatB/AnkB,KatG, TrxB, human MAP kinase phosphatase 1 (MKP-1) genes; mammalianhic-5 genes, and the isc operon.

In another embodiment the cell provided by the kit is a bacterial celland the reporter gene encodes a protein functional in bacteria. In apreferred embodiment, the bacterial cell is Agrobacterium tumefaciens.

In preferred embodiments of the method or kit of the invention, theROS-inducible promoter is from the KatA gene of Agrobacteriumtumefaciens.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Alignment of the amino acid sequence of the Agrobacteriumtumefaciens katA gene product with the homologous catalase sequences.Alignment was completed by using the DNAsis program. Atu KatA representsthe A. tumefaciens katA gene product; Lpn KatB represents the Legionellapneumophila katB gene product; Bst Cat represents the Bacillusstearothermophilus catalase.

FIG. 2. Catalase isozyme assays. A. Agrobacterium tumefaciens strainsA348 (lane 1), AG6 (pXQ9) (lane 2) and AG6 (lane 3) were grown overnightat 28° C. in MG/L liquid medium. Crude cell extracts were prepared asdescribed in the Materials and Methods. For each strain, 20 μl of crudecell extract was loaded and electrophoresed on 7.5% nondenaturing gel.B. 40 μl (lane 1) and 20 μl (lane 2) of A348 cell crude extract (thathad been diluted 2× further after the dilution described in theMaterials and Methods) was loaded and electrophoresed. Catalase isozymeswere visualised by activity staining according to Clare et al. (1984).

FIG. 3. The katA-gfp expression in different growth media. Agrobacteriumtumefaciens strains A348 and AG6 were grown at 28° C. for 24 hr on agarplates of MG/L, AB, IB (pH 5.5), and IB (pH 7.0) and fresh Kalanchoeleaf tissue and stem tissue sections. The cells were harvested and thenresuspended in dH₂O. The fluorescence of each cell suspension wasmeasured by Luminescence Spectrometer LS50B (Perkin Elmer) as describedin the Materials and Methods using A348 as the blank.

FIG. 4. Comparison of katA-gfp expression in different geneticbackgrounds. Agrobacterium tumefaciens strains A348, AG6, AG6 (pSW172),AG6 (pXQ9), AG613, CGI1 and CGI1 (pXQ9) were grown at 28° C. for 24 hron agar plates of IB (pH 5.5). The cells were harvested and thenresuspended in dH₂O. The fluorescence of each cell suspension wasmeasured by Luminescence Spectrometer LS50B (Perkin Elmer) as describedin the Materials and Methods using A348 as the blank.

FIG. 5. Schematic presentation of the wild-type and mutated katA genes.The lines represent the DNA sequences; the boxes represent the KatA openreading frames (ORFs). The vertical lines indicate the restriction sitesor amino acid positions. The diamond indicates the mini-Tn5 transposoninsertion position. The key restriction endonuclease sites and primersused are indicated. The DNA sequences under the triangles are the ORFsequences concerned for the site-directed mutagenesis. ˜ represents adeletion of the G of the second codon; * represents the stop codonintroduced at the fifth codon. The wild type katA in pXQ9 and the mutantkatA genes encoding KatAΔ50 and KatAΔ86 were driven by the katApromoter. The wild type katA in pXQ23 and the mutant genes encoding KatA(98H/D), KatA (94R/Q) (98H/D), ΔkatA (˜2) and ΔkatA (*5) were driven byboth the katA and lac promoter.

FIG. 6. The effects of katA mutations on the KatA protein stability.Agrobacterium tumefaciens strains A348 (panel A, lane 2; panel B, lane1), AG6 (panel A, lane 3), AG6 (pXQ23) (panel A, lane 4), AG6 (pXQ26)(panel A, lane 5), AG6 (pXQ27) (panel A, lane 6), AG6 (pXQ30) (panel A,lane 7), AG6 (pXQ31) (panel A, lane 8), AG6 (pXQ9) (panel B, lane 2),AG6 (pXQ11) (panel B, lane 3), and AG6 (pXQ22) (panel B, lane 4) weregrown overnight at 28° C. on IB plates. The cells were harvested, washedand diluted to a concentration of OD₆₀₀=0.3. The cells from 500 μl ofcell suspensions were harvested by centrifugation and resuspended in theLaemmli (1970) sample buffer. An aliquot of 2 μl of each sample waselectrophoresed on SDS/10% PAGE gels. The proteins were transferred ontoImmobilon-P membrane and visualized by (His)₆-KatA antibody. Thepurified (His)₆-KatA was used as the control.

FIG. 7. Detection of the GFP protein expression of the katA-gfp fusion.Agrobacterium tumefaciens strains A348 (lane 1), AG6 (lane 2), AG6(pSW172) (lane 3), AG6 (pXQ23) (lane 4), AG6 (pXQ26) (lane 5), AG6(pXQ27) (lane 6), AG6 (pXQ30) (lane 7), AG6 (pXQ31) (lane 8), AG6(pXQ11) (lane 9), and AG6 (pXQ22) (lane 10) were grown overnight at 28°C. on IB plates. The cells were harvested and then resuspended in dH₂O.One portion of each cell suspension was used to measure the fluorescenceby Luminescence Spectrometer LS50B (Perkin Elmer) as described in theMaterials and Methods using A348 as the blank (upper panel). Anotherportion of each cell suspension was diluted to a concentration ofOD₆₀₀=0.3. The cells from 500 μl of cell suspensions were harvested bycentrifugation and resuspended in the Laemmli (1970) sample buffer. Analiquot of 2 μl of each sample was electrophoresed on SDS/15% PAGE gels.The proteins were transferred onto Immobilon-P membrane; the GFP wasvisualized by the GFP antibody (lower panel).

FIG. 8. Assays for catalase activity bands. Agrobacterium tumefaciensstrains A348 (lane 1), AG6 (lane 2), AG6 (pXQ23) (lane 3), AG6 (pXQ26)(lane 4), AG6 (pXQ27) (lane 5), AG6 (pXQ11) (lane 6), and AG6 (pXQ22)(lane 7) were grown overnight at 28° C. in MG/L liquid medium. Samplesof crude cell extracts were prepared and electrophoresed on 7.5%nondenaturing gel as described previously (Xu and Pan, 2000). Catalaseisozymes were visualized by activity staining according to Clare et al(1984). Since AG6 (pXQ23) was over-expressing KatA, this sample wasdiluted 8 fold before loading.

FIG. 9. Induction of the katA-gfp fusion by H₂O₂. The cells ofAgrobacterium tumefaciens AG6 grown in MG/L (OD₆₀₀=0.5) were exposed to0, 30 μM, 60 μM, and 120 μM H₂O₂. The cell suspensions were incubated at28° C. for 2 hours. Aliquots of 1 ml cell cultures were harvested bycentrifugation and resuspended in the Laemmli (1970) sample buffer. Analiquot of 10 μl of each sample was electrophoresed on SDS/15% PAGEgels. The proteins were transferred onto Immobilon-P membrane; the GFPwas visualized by the GFP antibody.

FIG. 10. Repression of katA-gfp expression by surrounding bacterialcells. The AG6 cells were mixed with at 1:1 ratio with the cells fromthe bacterial strains A348, Rhizobium meliloti RCR2011, or E. coli DH5α;the mixtures were spotted on IB plates. The same amount of bacterialcells from a single strain A348, AG6 (pXQ9), Rhizobium meliloti RCR2011,or E. coli DH5α was also spotted on IB plates. The plates were incubatedovernight at 28° C. The bacterial fluorescence under UV light wasphotographed (upper panel). The fluorescence intensity was measured(lower panel) as described in the Materials and Methods.

DETAILED DESCRIPTION OF THE EMBODIMENTS

To determine whether a compound is able to remove an ROS, anROS-inducible promoter (RIP) is fused to a reporter gene to drive itsexpression. The reporter gene is heterologous to the promoter to whichit is operably linked. The RIP-reporter construct is then stablytransformed into the cell. To test whether a certain compound is anROS-remover, the cell is exposed to the test compound. Preferably, thecell is exposed to the test compound intracellularly. If necessary, theintracellular level of the ROS is induced, and the ROS-inducibleexpression level of the reporter gene in the cell is measured. Areduction in the reporter protein level when the cell is exposed to thecompound would indicate that the compound is able to remove the ROS.

The test compound may also be provided by being expressed in aneighbouring cell, rather than being expressed from the cell containingthe RIP.

As used herein, the terms “reactive oxygen species” (ROS) and “oxidants”are used interchangeably, and include hydroxyl radicals, superoxideanion, hydrogen peroxide and nitric oxide.

ROS-removing compounds are anti-oxidants or oxidant scavengers. Namely,these compounds have the ability remove ROS by breaking them downchemically, or by sequestering them away from solution. KnownROS-removing compounds include proteins, such as ferritin, lactoferritinand transferritin, or enzymes, such as superoxide dismutase, catalaseand glutathione peroxidase. Nonenzymatic antioxidants may bemacromolecules, such as albumin, copper-binding ceruloplasmin andhemoglobin, or small molecules such as water-soluble antioxidants (e.g.vitamin C, uric acid and bilirubin) or lipid-soluble antioxidants (e.g.vitamin E, carotenoids, retinoids and ubiquinol-10).

In a preferred embodiment, the test compounds with potentialROS-removing capability are proteins expressed intracellularly. They areoften proteins heterologous to the cells containing the RIP-reporterconstruct. However, a polypeptide naturally present in such cells mayalso be tested as a ROS-removing compound provided that the geneencoding the functional polypeptide has been knocked out from the cell.

The term “heterologous” means, in the context of the present invention,that the components are not found naturally together. For example, areporter gene which is heterologous to the promoter to which it islinked is not the natural coding sequence of the gene from which thepromoter is derived.

The term “vector” refers to a nucleic acid sequence that is capable ofpropagating in particular host cells and can accommodate inserts offoreign nucleic acid. Typically, vectors can be manipulated in vitro toinsert foreign nucleic acids and the vectors can be introduced into hostcells such that the inserted nucleic acid is transiently or stablypresent in the host cells.

The term “expression vector” refers to a vector designed to expressinserted nucleic acid sequences. Such vectors may contain a powerfulpromoter located upstream of the insertion site.

The term “expression” in the context of nucleic acids refers totranscription and/or translation of nucleic acids into mRNA and/orprotein products.

The term “expression library” refers to a library of nucleic acidfragments contained as inserts in an expression vector.

The term “stable transformation” refers to the continued presence of anucleic acid sequence in a host cell for a period of time that is atleast as long as that required to carry out the methods of the presentinvention. Stable transformation can be achieved through integration ofthe construct into a host cell chromosome, or engineering the constructso that it possesses elements that ensure its continued replication andsegregation within the host (i.e., an artificial chromosome), oralternatively, the construct may contain a selectable marker (e.g., adrug resistance gene) so that persistence of the construct in the cellis ensured by growing the host cells under selective conditions (e.g.,in drug-containing media).

The term “cell” or “host cell” in the present invention refers to a cellof prokaryotic or eukaryotic origin that can serve as a recipient of anintroduced vector. The host cell often allows replication andsegregation of the vector that resides within. In certain cases,however, replication and/or segregation are irrelevant; expression ofvector or insert DNA is the objective. Typical bacterial host cellsinclude E. coli, B. subtilis and A. tumefaciens; fungal host cellsinclude S. cerevisiae and S. pombe; plant cells include those isolatedfrom A. thaliana, and Z. maize; insect host cells include those isolatedfrom D. melanogaster, A. aegypti, and S. frugiperda; and mammalian cellsinclude those isolated from human tissues and cancers includingmelanocyte (melanoma), colon (carcinoma), prostate (carcinoma), brain(glioma, neuroblastoma, astrocytoma) and liver (hepatoma).

An ROS-inducible promoter is one which, in response to the presence ofthe ROS inside the cell, expression from the promoter is increased.Numerous ROS-inducible promoters are known in the art. They include:AhpCF, Bcp, Dps, gor, KatA, KatB/AnkB, KatG, TrxB, human MAP kinasephosphatase 1 (MKP-1) genes; mammalian hic-5 genes, the isc operon;Escherichia coli zwf, fpr, fumC, micF, nfo, and sodA genes; Azotobactervinelandii spr gene; Xanthomonas oryzae pv oryzae katX gene; rat andhuman haem oxygenase-1 (HO-1); yeast 2-deoxyglucose-6-phophatephosphatase (DOG2); catalase; human manganese superoxide dismutase(MnSOD); rat glutathione S-transferase (GST); human interstitialcollagenase (MMP-1); human glutathione peroxidase (GPX2); fishmetallothionein (MT); and rat multidrug resistance type 1 (mdr1).

It is expected that most of the promoters above would be functional tosome degree in a heterologous cell type. However, it is preferred thatpromoters naturally found in bacteria would be used in bacteria, andyeast promoter in yeast cells, and mammalian promoters in mammaliancells, according to the methods of the invention.

In a preferred embodiment, the ROS is H₂O₂, and the H₂O₂-induciblepromoter is from a gene such as AhpCF, Bcp, Dps, gor, KatA, KatB/AnkB,KatG, TrxB, human MAP kinase phosphatase 1 (MKP-1) genes; mammalianhic-5 genes, and the isc operon.

The RIP-reporter vector is customized so that reporter expressionreflects as closely as possible the ROS level of the host cell. Thus,the expression vector is designed so that the reporter gene is placedunder control of ROS-response cis regulatory elements functional in thehost cell. Preferably, the reporter is expressed at a low level in theabsence of the ROS; i.e. the basal activity of the promoter should below so that induction by ROS is readily detectable.

A partial listing of the genes, their organism of origin, and Genbankaccession numbers are provided below. A brief description of some ofthese genes and reference publications are also provided:

-   -   Streptococcus mutans ahpC and nox1 genes for alkyl        hydroperoxidase and NADH oxidase/alkyl hydroperoxidase        reductase, ACCESSION AB010712.

Mycobacterium marinum alkylhydroperoxide reductase (ahpC) gene,ACCESSION AF034861.

-   -   Bacteroides fragilis alkyl hydroperoxide reductase subunit C        (ahpC) and alkyl hydroperoxide reductase subunit F (ahpF) genes,        ACCESSION AF129406.    -   Salmonella typhimurium alkyl hydroperoxide reductase (ahpC) and        (ahpF) genes, ACCESSION J05478.    -   Mycobacterium avium alkyl hydroperoxidase C (ahpC) gene,        ACCESSION U18263.    -   Mycobacterium tuberculosis alkyl hydroperoxidase C (ahpC) gene,        ACCESSION U18264.    -   Mycobacterium smegmatis alkyl hydroperoxide reductase C (ahpC)        gene, ACCESSION U43719.    -   Mycobacterium intracellulare alkyl hydroperoxidase C (ahpC),        ACCESSION U71061.    -   Staphylococcus aureus alkyl hydroperoxide reductase subunit C        (aphC) and subunit F (aphF) genes, ACCESSION U92441 X85029.

Escherichia coli bacterioferritin comigratory protein (bcp), ACCESSIONM63654 M37689.

-   -   Escherichia coli DNA binding protein Dps (dps) gene, ACCESSION        AF140030.    -   Bacteroides fragilis non-specific DNA-binding protein Dps (dps),        ACCESSION AF206033.    -   Synechococcus sp. nutrient-stress induced DNA binding protein        (dpsA) gene, ACCESSION U19762.    -   Streptococcus thermophilus glutathione reductase (gor) gene,        ACCESSION L27672.    -   E.coli gor gene encoding glutathione reductase, ACCESSION        M13141.    -   P. aeruginosa gor gene for glutathione reductase (EC 1.6.4.2),        ACCESSION X54201.    -   Agrobacterium tumefaciens catalase (KatA), SEQ ID NO:1.    -   Vibrio fischeri catalase (katA) gene, ACCESSION AF011784.    -   Pseudomonas aeruginosa catalase isozyme A (katA) gene, ACCESSION        AF047025.    -   Actinobacillus actinomycetemcomitans catalase (katA) gene,        ACCESSION AF162654.    -   Legionella pneumophila catalase-peroxidase (katA) gene,        ACCESSION AF276752.    -   Staphylococcus aureus catalase gene, strain ATCC12600. ACCESSION        AJ000472.    -   Lactobacillus sake catalase (katA) gene, ACCESSION M84015.    -   Rhizobium meliloti catalase (katA) gene, ACCESSION U59271.    -   Pseudomonas fluorescens plasmid pAM10.6 catalase isozyme (katA)        ACCESSION U72068.    -   H.pylori katA gene, ACCESSION Z70679.    -   B.subtilis 25 kb genomic DNA segment (from sspE to katA),        ACCESSION Z82044.    -   Pseudomonas aeruginosa paraquat inducible catalase isozyme B        (katB), ankyrin (ankB), ACCESSION U89384.    -   Caulobacter crescentus catalase-peroxidase (katG) gene,        ACCESSION AF027168.    -   Mycobacterium smegmatis catalase-peroxidase (katG) gene,        ACCESSION AF196484.    -   Synechococcus PCC6301 catalase-peroxidase gene, ACCESSION        AF197161.    -   Mycobacterium leprae DNA for catalase-peroxidase, ACCESSION        D89336.    -   E.coli katG gene encoding catalase HP1, ACCESSION M21516.    -   Salmonella typhimurium Kat G gene for hydroperoxidase I.        ACCESSION X53001.    -   M.tuberculosis katG gene for catalase-peroxidase. ACCESSION        X68081 S42739.    -   M.bovis katG gene. ACCESSION X83277.    -   M.smegmatis katG gene. ACCESSION X98718.    -   M.fortuitum katGI gene. ACCESSION Y07865.    -   M.fortuitum katGII gene. ACCESSION Y07866.    -   Mycobacterium smegmatis thioredoxin reductase (trxB) and        thioredoxin (trxA) genes, ACCESSION AF023161.    -   Streptomyces coelicolor sigT, trxB and trxA genes, ACCESSION        AJ007313.    -   Clostridium litorale thioredoxin reductase (trxB), and        thioredoxin (trxA) genes, ACCESSION U24268.    -   Mycoplasma pneumoniae thioredoxin reductase K04_orf315 (trxB)        gene, ACCESSION U51988.    -   The sodA gene encodes superoxide dismutase and is strongly        induced when cells are exposed to chemicals that produce        superoxide radicals in the cell, such as paraquat, plumbagin,        menadione, streptonigrin, methylene blue and phenazine methyl        sulfate. SodA gene induction depends upon an increase in steady        state superoxide concentration, not necessarily upon cellular        damage caused by superoxides.    -   The soi28 gene encodes a pyruvate:flavodoxin oxidoreductase.        This gene is induced by superoxide-producing reagents only.        Specifically, the soi28 gene is induced when two small,        thiol-containing proteins, flavodoxin and ferredoxin, become        oxidized.    -   The ahp gene is induced by hydrogen peroxide and organic        hydroperoxides, both exogenous and those formed upon        peroxidation of proteins and fatty acids.    -   soi17 and soi19 respond to superoxides [T. Kogoma et al.,        (1988)].    -   zwf encodes glucose-6-hydrogenase and is induced by        superoxide-producing compounds and nitric oxide [Greenberg et        al.(1990)].    -   micF encodes antisense RNA that shuts off translation of the        porin gene, ompF and is induced by superoxides [Greenberg et        al.(1990)].    -   The nfo gene encodes a DNA repair enzyme and is specifically        induced by redox active agents, such as paraquat and menadione        [Farr et al.(1991)].

If the nucleotide sequence of the ROS-inducible gene is known,polymerase chain reaction may be used to produce fusions with thepromoter. Specifically, primers are synthesized which are complementaryto the 5′ and 3′ ends of the ROS- inducible promoter portion of thegene, hybridizes those primers to denatured, total DNA under appropriateconditions and performs PCR. In this manner, clonable quantities of anysequenced promoter may be obtained. Once the promoter DNA has beenobtained, it is ligated to a DNA encoding the reporter gene in anappropriate vector, such as pRS415 for E. coli, which contains amultiple cloning site just upstream from the lacZ gene. Numerous vectorsfor expressing reporter genes are known in the art or are commerciallyavailable. The methods are well-known in the art.

A reporter gene as used in the present invention essentially encodes anygene product that can be expressed in the cell of interest and isassayable and detectable. The reporter gene must be sufficientlycharacterized such that it can be operably linked to the promoter.Reporter genes used in the art include the LacZ gene from E. coli(Shapiro S. K., Chou J., et al., Gene November; 25: 71-82 (1983)), theCAT gene from bacteria (Thiel G., Petersohn D., and Schoch S., GeneFebruary 12; 168: 173-176 (1996)), the luciferase gene from firefly(Gould S. J., and Subramani S., 1988), the GFP gene from jellyfish(Chalfie M. and Prashner D. C., U.S. Pat. No. 5,491,084), galactosekinase (encoded by the galK gene), and beta-glucosidase (encoded by thegus gene). These have been primarily used to monitor expression of genesin the cytoplasm. To monitor expression at the cell surface, a labeledantibody that binds to the cell surface marker (e.g., CD20) may be usedto quantify the level of reporter (Koh J., Enders G. H., et al, 1995).

Of these reporters, autofluorescent proteins (e.g., GFP) and the cellsurface reporters are preferred for use in monitoring living cells,because they act as “vital dyes”. Their expression can be evaluated inliving cells, and the cells can be recovered intact for subsequentanalysis. Vital dyes, however, are not specifically required by themethods of the present invention. It is also very useful to employreporters whose expression can be quantified rapidly and with highsensitivity. Thus, fluorescent reporters (or reporters that can belabeled directly or indirectly with a fluorophore) are especiallypreferred. This trait permits high throughput screening on a flow sortermachine such as a fluorescence activated cell sorter (FACS).

GFP is a member of a family of naturally occurring fluorescent proteins,whose fluorescence is primarily in the green region of the spectrum. GFPhas been developed extensively for use as a reporter and several mutantforms of the protein have been characterized that have altered spectralproperties. High levels of GFP expression have been obtained in cellsranging from yeast to human cells. It is a robust, all-purpose reporter,whose expression in the cytoplasm can be measured quantitatively using aflow sorter instrument such as a FACS.

The diagnostic kits and methods of this invention rely on the inductionof specific ROS-inducible promoters to alter expression of the reportergene. This change in expression level is measured both qualitatively andquantitatively. In order to be useful in those kits and methods, theparticular stress promoter must be operably linked to the gene whichencodes the reporter product.

The term “operable linkage” refers to the positioning of the promoterrelative to the gene encoding the reporter product such thattranscription of the gene is regulated by the promoter. Such positioningis well known in the art and involves positioning the promoter upstream(5′) of the gene so that transcription is not impeded by extraneoustermination signals and where the spacing between the promoterinitiation site and the regulatory sequences of the promoter are optimalfor transcription.

Also within the scope of this invention are constructs wherein thereporter product is in fusion with the N-terminal portion of the nativegene product, i.e. the gene product of the promoter to which thereporter is fused. It is important that the portion of native geneproduct fused to the reporter does not retain the function of the fulllength native gene product.

The choice of bacterial strain to express the particular RIP-reporterconstruct and thus useful in the methods and kits of this invention isonly limited by the strain's ability to produce the functional reporterand its inability to synthesize the reporter in its untransformed state.Most preferably, the strain used should be defective in genes whichendogenously remove ROS intracellularly. Such genes include thoseencoding catalase, superoxide dismutase, alkyl hydroperoxidase, andglutathione reductase. For example, where an H₂O₂-inducible promoter isused, it is preferred that the endogenous catalase genes be knocked outor mutated in the cells so that the cells lose or have decreasedcapacity to break down H₂O₂ endogenously.

Eukaryotic cells useful in the methods and kits of the invention includecell lines established from primary tissue, as well as those cell linesand cultures available from the American Type Culture Collection (ATCC,Rockville, Md.).

The method and kits of the invention rely on a detectable reduction inROS-inducible reporter expression to test whether a compound is capableof removing ROS. This requires that the level of reporter expression besufficiently high in the absence of an ROS-removing compound, so that areduction is detectable. In some embodiments, the method involveselevating the intracellular level of the ROS. Methods used to elevatethe concentration of various intracellular ROS are known.

In bacteria, intracellular levels of H₂O₂ may be elevated by usingglucose/glucose oxidase (GOX) or reduced glutathione (GSH) asH₂O₂-generating systems (Saliim et al. 2001). In Agrobacterium,intracellular levels of H₂O₂ may be elevated by acid conditions. Inmammalian cells and in yeast, depletion of intracellular glutathioneraises intracellular ROS. In at least mammalian cells, glutathione maybe depleted by application of buthionine sulfoximine. Insulinestimulation also generates a burst of intracellular H₂O₂ ininsulin-sensitive hepatoma and adipose cells (Mahadev et al. 2001). InArabidopsis, application of dexamethasone activates MAP kinases andresults in the generation of H₂O₂ (Ren et al. 2002).

In other embodiments, the method involves using cells where the cellshave been genetically modified so that there is an elevatedintracellular level of an ROS. In bacteria, E. coli strains wheremodulation of expression of superoxide dismutase results in modulationof intracellular superoxide (Gort and Imlay, 1998). In yeast, expressionof cytochrome peroxidase, superoxide dismutase or the GSH1 gene may bemodulated. In fibroblasts, cells that stably express Nox1 produces amarked increase in intracellular H₂O₂, as well as some increase insuperoxide level (Arnold et al. 2001).

In an exemplary embodiment, an assay testing for a compound for itsability remove H₂O₂ would proceed along the following line. Anexpression construct which expresses a potential H₂O₂-remover isintroduced into a cell line which contains a reporter gene under controlof an H₂O₂-inducible promoter, such as the A. tumefaciens strain AG6.Production of intracellular H₂O₂ may be induced, for example by exposingthe cells to low pH medium. The level of reporter protein, as indicatedby the level of fluorescence if the reporter is GFP, would be reduced inthe cells expressing an H₂O₂-removing compound, compared to the controlcells in which the compound is absent.

An aspect of the invention relates to a method for selecting a nucleicacid which encodes a protein potentially able to remove an ROS. In thismethod, cells are provided which contain the RIP-reporter gene constructas described above. Expression vectors containing different nucleicacids, such as those found in a cDNA library, or in a library where thenucleic acids have been mutagenized, are used to transform the cells.These nucleic acids encode proteins which are potentially able to removethe ROS. Any reduction in the ROS-inducible expression of the reportergene is measured, as described above, when the nucleic acids areexpressed. The cells with reduced ROS-inducible expression of thereporter gene are then selected and the nucleic acid used to transformthe cell is isolated. This nucleic acid would likely encode anROS-removing protein.

The term “library” refers to a collection of nucleic acid fragments thatmay individually range in size from about a few base pairs to about amillion base pairs. These fragments are contained as inserts in vectorscapable of propagating in certain host cells such as bacterial, fungal,plant, insect, or mammalian cells.

The term “plurality of nucleic acids” refers to a set of nucleic acidmolecules from any source. For example, a plurality of nucleic acids maycomprise total genomic DNA, genomic DNA from one or more chromosomes,cDNA that has been reverse-transcribed from total cellular RNA or frommessenger RNA (mRNA), total cellular RNA, mRNA, or a set of nucleic acidmolecules synthesized in vitro either individually, or usingcombinatorial methods. Plurality of nucleic acids is understood toinclude, e.g. an expression library.

The terms “bright” and “dim” in the context of a cell sorter refer tothe intensity levels of fluorescence (or other modes of light emission)exhibited by particular cells: Bright cells have high intensity emissionrelative to the bulk population of cells, and by inference, high levelsof reporter gene expression; dim cells have low intensity emissionrelative to the bulk population.

The term “flow sorter” refers to a machine that analyzes light emissionintensity from cells or other objects and separates these cells orobjects according to parameters such as light emission intensity.

In one embodiment, the method using GFP as a reporter protein, to selectfor ROS-removing proteins is as follows. The ROS-inducible promoter-GFPexpression construct is introduced into the chosen host cells and astable expresser is selected. This GFP-expressing line is clonallyexpanded to generate a population that is bright green. A libraryencoding potential ROS-removers is introduced into the host cells togenerate a population of GFP-containing cells, some of which alsoexpress ROS-removers. This population is examined using a flow sorterdevice and cells are sorted into two populations: cells that continue toexpress GFP at levels similar to the cells before introduction of thelibrary inserts.; and, cells that express reduced levels of GFP. Theinserts encoding ROS-removers from such “dim” cells are isolated andeither used to determine their DNA sequences, or reintroduced into theGFP-containing host cells for another cycle of selection and enrichment.

One can envision a flow sorter profile diagram of the selectionprocedure described above. The fluorescence intensity of a population ofhost cells containing the library inserts prior to selection would havea normal distribution. This presorted population is used to select cellson the left tail of the distribution. The dim cells on the left of thedistribution are selected and inserts from these cells are reintroducedinto the original host cells. The fluorescence intensity distributionthat ensues from cells transformed with such a sub-library of sequenceswould become skewed to the left (i.e., the mean fluorescence intensitydecreases).

The present invention may use a flow sorter such as a FACS or equivalentdevice to screen through large numbers of host cells containingexpression library inserts encoding potential ROS-removing proteins, toidentify those that can remove ROS; namely, cells that have reducedlevels of reporter molecule expression. Host cells which have anelevated level of ROS and which have the reporter (e.g., GFP) presentunder control of the ROS-inducible promoter will have a constitutivelyhigh level of reporter expression. When the expression library insertsare expressed in these cells, the large majority of cells that areanalyzed by FACS are expected to have retained this high level. However,a small number may exhibit reduced expression, detected on the FACS ascells that fall on the dimmer side of the cell fluorescencedistribution. These dim cells can be collected and grown in isolation ofthe others. Such a procedure results in enrichment from the startingpopulation of cells for those that contain ROS-removers, whicheffectively reduce the level of inducer ROS, thereby reducing the levelof reporter expression. These selected, dim cells can be used toreisolate the perturbagen fragments by, e.g., PCR using primer sitesthat flank the library inserts, so as to build a sub-library of libraryinserts enriched for those that cause reduced reporter expression. Thesub-library of fragments can be recloned (using e.g., the sameexpression vector) and reintroduced into the host cells, and thescreening/selection process can be repeated as many times as necessary.

After a sufficient number of cycles, a substantial difference should beobserved in the fluorescence intensity distribution of the originalreporter-containing host cells as compared to the host cells harboringthe enriched ROS-removing sub-library inserts. Preferably, the procedureshould be repeated until a minimal overlap is observed between these twofluorescence intensity distributions. Ultimately, the process of FACSsorting and cycling should result in a population of nucleic acidsencoding ROS-removers that inhibit expression of the reporter. These canbe isolated and studied individually by molecular cloning and DNAsequence analysis.

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting this invention in any manner.

EXAMPLE 1 Materials, Techniques and Assay Conditions

Strains, plasmids, and growth conditions: The strains and plasmids usedin this study are listed and described in Table 1. Agrobacteriumtumefaciens strains were grown in MG/L, IB or AB medium (Cangelosi etal., 1991) at 28° C., supplemented with 100 μg/ml kanamycin, 5 μg/mltetracycline, or 100 μg/ml carbenicillin as required. Escherichia colistrains were grown on Luria-Bertani (LB) medium (Sambrook et al., 1989)at 37° C., supplemented with 50 μg/ml kanamycin, 10 μg/ml tetracycline,or 50 μg/ml ampicillin as required. Mini-Tn5 transposon mutagenesis ofA. tumefaciens strain A348 was carried out by pAG408 as described(Suarez et al., 1997).Southern analysis: Total DNA was extracted as described previously(Charles and Nester, 1993) from the A. tumefaciens mutant AG6.Approximately 1 μg of total DNA was digested with ClaI or NruI and thenelectrophoresed on a 0.9% agarose gel. The DNA fragments were thentransferred onto nylon membrane Zeta-Probe GT (Bio-Rad) using a transferapparatus, PosiBlot (Stratagene). The plasmid pAG408 was labelled as theprobe by random priming with the enhanced chemiluminescence kit(Amersham). The labelling, hybridisation and signal detection wereconducted according to the manufacturer.Catalase isozyme assay: A. tumefaciens strains A348, AG6, AG6 (pXQ23),AG6 (pXQ26), AG6 (pXQ27), AG6 (pXQ11), and AG6 (pXQ22) were grownovernight at 28° C. in MG/L liquid medium to 1.4 OD₆₀₀. The cells wereharvested by centrifugation at 4000 rpm for 10 min at 4° C. The cellpellets were washed and resuspended in 5 ml extraction buffer containing0.05 mM phosphate and 0.4 mM EDTA (pH 7.8). The cells were sonicated for30 sec for 6 times on ice with a 2-min cooling on ice betweensonications. The cell debris was removed by centrifugation at 1100 rpmfor 10 min at 4° C. The cell-free supernatant was diluted 2× with theextraction buffer, and 20 μl of each diluted extract was electrophoresedon 7.5% native polyacrylamide gels. The resolving gel buffer wasprepared at pH 8.1 instead of pH 8.9. Electrophoresis was performed at150 V for 3 hours. Catalase isozymes were visualised by an activitystaining procedure according to Clare et al. (1984).Protein analysis: SDS/PAGE was conducted in 10% or 15% polyacrylamidegels to analyze the KatA or GFP expression, respectively. The proteinswere transferred onto Immobilon-P membranes (Millipore). The KatA or GFPproteins were visualized with the enhanced chemiluminescence (ECL)western blot detection system according to the recommendations of themanufacturer (Amersham).

TABLE 1 Bacterial strains and plasmids used in this study Source/Strain/plasmid Relevant characteristics* Reference Strains Agrobacteriumtumefaciens A348 Wild type, A136(pTiA6NC) (octopine-type) Laboratorycollection AG6 Derivative of A348 in which katA was disrupted by theThis study GFP-tagged mini-Tn5 transposon at 995 bp downstream from thestart condon of katA; Km^(R), Gm^(R) AG613 AG6 containing pXQ13integrated into the This study chromosome (containing a single copy ofthe wild type katA and katA-gfp fusion); KmR, Gm^(R), Cb^(R) CGI1Derivative of C58 in which aopB was disrupted by the This studyGFP-tagged mini-Tn5 transposon; Km^(R), Gm^(R) Escherichia coli DH5asupE Δlac(Δ80ZΔM15) hsdR recA endA gyrA thi relA Bethesda ResearchLaboratories MT607 Pro-82 thi-1 hsdR 17 supE44 endA1 recA56 Finan, etal., 1986 Plasmids pTZ19R Cloning vector, ColE1 oriV bla, Amp^(R) USBiochemical pSW172 Broad-host-range IncP plasmid containing P_(lac) andChen and downstream polylinker sequence, Tc^(R) Winans, 1991 pXQ6pBluescript II KS(−) containing a 6-kb ClaI DNA This study fragmentcontaining the sequences downstream of the mini-Tn5 insertion at thekatA gene. pXQ7 pTZ19R containing a 5-kb NruI DNA fragment This studycontaining the sequences upstream of the mini-Tn5 insertion at the katAgene. pXQ9 pSW172 carrying a 2.8 kb XbaI-NheI fragment This studycontaining the wild type katA, Tc^(R) pXQ11 pSW172 carrying a 2.3 kbXbaI-NheI fragment This study containing a KatA with a 86 amino aciddeletion at the C-terminus, Tc^(R) pXQ13 pTZ19R carrying a 2.8 kb EcoRIfragment from pXQ9 This study containing the wild type katA, Amp^(R)pXQ15 pRSETA carrying a 2.17 kb XhoI-KpnI fragment This study containingthe full length KatA ORF fused in-frame with (His)₆, Amp^(R) pXQ22pSW172 carrying a 2.4 kb XbaI-NheI fragment This study containing a KatAwith a 50 amino acid deletion at the C-terminus, Tc^(R) pXQ23 pSW172ligated with pXQ13 at ClaI, containing the This study wild type katA,Amp^(R) pXQ24 pTZ19R carrying a 2.8 kb EcoRI fragment from pXQ9 Thisstudy containing a katA with His 98 replaced by Asp, Amp^(R) pXQ25pTZ19R carrying a 2.8 kb EcoRI fragment from pXQ9 This study containinga katA with Arg 94 replaced by Gln and His 98 replaced by Asp, Amp^(R)pXQ26 pSW172 ligated with pXQ24 at ClaI, containing a katA This studywith His 98 replaced by Asp, Amp^(R), Tc^(R) pXQ27 pSW172 ligated withpXQ25 at ClaI, containing a katA This study with Arg 94 replaced by Glnand His 98 replaced by Asp, Amp^(R), Tc^(R) pXQ28 pTZ19R carrying a 2.8kb EcoRI fragment from pXQ9 This study containing the a katA with Ser5replaced by a stop codon (TGA), Amp^(R) pXQ29 pTZ19R carrying a 2.8 kbEcoRI fragment from pXQ9 This study containing the a katA with a G basepair deletion in the second codon of katA ORF, Amp^(R) pXQ30 pSW172ligated with pXQ29 at ClaI, containing a katA This study with a G basepair deletion in the second codon of katA ORF, Amp^(R), Tc^(R) pXQ31pSW172 ligated with pXQ28 at ClaI, containing a katA This study withSer5 replaced by a stop codon (TGA), Amp^(R), Tc^(R)Km, kanamycin; Tc, tetracycline; Amp, ampicillin; Gm, gentamycin.Measurement of intracellular H₂O₂ concentrations: The intracellularconcentrations of H₂O₂ were measured by the procedures previouslydescribed (González-Flecha and Demple, 1995; 1997) with modifications.

Briefly, A. tumefaciens strains A348, AG6 and AG6 (pXQ9) were grown at28° C. for 24 hr on agar plates of AB or IB. The cells were harvested,washed and resuspended at OD₆₀₀=1.0 in 50 mM phosphate-buffer (pH 7.4).H₂O₂ generated within the cells passed through membranes andequilibrated with the buffer. Complete equilibration of theintracellular and extracellulare H₂O₂ levels occurred within 10 min inthe assay (González-Flecha and Demple, 1997). After 20 min ofequilibration, the cell suspensions were centrifuged for 1 min at 6,000rpm at 4° C.

H₂O₂ concentrations in the supernatants were then measured by the AmplexRed Hydrogen Peroxide Assay Kit (Molecular Probes Inc., USA), whichcontains a highly sensitive and specific fluorogenic probe(N-acetyl-3,7-dihydroxyphenoxazine) for H₂O₂ and horse radish peroxidase(HRP) (Mohanty, et al, 1997). Briefly, 100 μl supernatant was mixed with100 μl of the probe at 100 μM and 1 U/ml HRP. The fluorometric assay wasconducted in a 96-well microplate and measured by LuminescenceSpectrometer LS50B (Perkin Elmer). The excitation wavelength was 540 nm;the emission wavelength was 590 nm. The assays were run in fourreplicates; the concentrations were then calculated based on the H₂O₂standard curves generated simutaneously.

Catalase activity assay: The catalase activity in whole bacterial cellswas determined as previously described (Maciver and Hansen, 1996) exceptusing the Amplex Red Catalase Assay Kit (Molecular Probes Inc., USA).Briefly, A. tumefaciens strains were grown at 28° C. for 24 hr on IBplates. The cells were harvested, washed and resuspended at OD₆₀₀=1.0 in50 mM phosphate-buffer (pH 7.4). For the strains with the wild type katA[A348, AG6 (pXQ9), and AG6 (pXQ23)], 100 μl cell suspension of eachsample was incubated with 50 μl 40 μM H₂O₂ at time intervals of 0, 30sec, 1 min and 2 min.

For the strains with the katA mutants [AG6, AG6 (pXQ11), AG6 (pXQ22),AG6 (pXQ26) and AG6 (pXQ27)], 100 μl cell suspension of each sample wasincubated with 50 μl 5 μM H₂O₂ at time intervals of 0, 1, 2, and up to 6min. The amount of H₂O₂ left after degradation by the bacterial catalasewas then determined by adding 50 μl Amplex Red reagent N-acetyl-3,7-dihydroxyphenoxazine at 25 μM and 0.4 U/ml horse radish peroxidaseprovided by the kit. The fluorometric assay was conducted by the sameprocedure as described above for the measurement of intracellular H₂O₂.The cell suspensions without the added H₂O₂ were used as the blank.Solutions of crystalline bovine catalase (Sigma) were used tostandardize this assay.

EXAMPLE 2 Cloning, Sequencing and Characterization of the katA GeneEncoding Catalase from Agrobacterium

The total DNA was extracted from AG6 which contains a mini-Tn5 insertionat the katA gene. Southern analysis revealed a 6-kb ClaI DNA fragmentcontaining the sequences downstream of the mini-Tn5 insertion at thekatA gene and a 5-kb NruI DNA fragment containing the sequences upstreamof the insertion. Those DNA fragments were extracted from the agarosegels and were cleaned by using GENECLEAN II Kit (BIO 101). The ClaI DNAfragment was cloned into pBluescript II KS (−) at the ClaI site, and theNruI DNA fragment was cloned into pTZ19R at the SmaI site.

The resulting plasmids were designated as pXQ6 and pXQ7, respectively.Sequencing of pXQ6 and pXQ7 was carried out using a mini-Tn5 specificprimer and the M13 reverse and −40 universal primers. The resultingsequence data were then used to generate primers for further sequencing.DNA sequencing was carried out using the ABI PRISM 377 DNA Sequencer.

In order to clone the full length katA gene, primers p83(5′-GGTGCGCTAGCCAAATTCGTCACCAAGC-3′) and n84(5′-CAATCGCTAGCGTTCGGCCCTCTG-3′) were designed that can respectivelyreanneal to the upstream and downstream sequences of the katA gene. Bothprimers had a NheI site to facilitate subsequent cloning. The total DNAfrom A. tumefaciens strain A348 was used as the template for PCR toamplify a 2.9 kb DNA fragment. The PCR product was digested with NheIand ligated into pSW172 (Chen and Winans, 1991) that had been digestedwith XbaI. The resulting plasmid pXQ9 was sequenced in both directionsindependently to obtain unambiguous sequence data. Plasmid pXQ9 wasintroduced into the mini-Tn5 mutant AG6 to create AG6 (pXQ9) bytriparental mating (Ditta et al., 1980) based on selection on MG/Lmedium supplemented with 100 μg/ml of kanamycin and 5 μg/ml oftetracycline.

The following is a characterization of katA encoding catalase fromAgrobacterium. A. tumefaciens A348 was mutagenized with a mini-Tn5transposon containing a promoter-less gene encoding a green fluorescentprotein (GFP) variant, which produces bright green fluorescence under UVlight. The mini-Tn5 transposon was carried on a plasmid pAG408 (Suarezet al., 1997). One of the mutants AG6 contained the transposon insertionat a gene that was differentially induced by pH on a minimal medium.

The leaves of Kalanchoe plants were inoculated with this mutant strainAG6 and compared with the parent strain A348. AG6 was highly attenuatedin the ability to cause tumors on plants as compared with A348. In orderto isolate the mini-Tn5 containing DNA fragments, Southern analysis wasconducted to estimate their sizes. A 6-kb ClaI DNA fragment containingthe sequences downstream of the transposon insertion site and a 5-kbNruI DNA fragment containing the sequences upstream of the insertionsite were cloned into the vectors, resulting in plasmids pXQ6 and pXQ7,respectively. Sequence analysis of pXQ6 and pXQ7 revealed that thetransposon was inserted at a gene that is homologous to bacterial genesencoding catalases. This gene is designated as katA.

In order to determine the complete sequence of the gene, the DNAfragment was amplified from A348 by polymerase chain reaction (PCR). Afragment of 2.9 kb was obtained that contained both the upstream anddownstream sequences of katA. The resulting fragment was cloned intopSW172 (Chen and Winans, 1991) to generate plasmid pXQ9. When pXQ9 wasintroduced into AG6, it could fully restore the ability of the mutant tocause tumors, suggesting that pXQ9 carried a full length katA gene.Sequence analysis indicated that the katA locus carried a single openreading frame (ORF) which encodes a putative protein of 723 amino acidswith a molecular weight of 78.7 kDa. This putative protein was highlyhomologous to other bacterial catalases (FIG. 1).

To determine whether the katA gene encodes a functional catalase, thecatalase isozyme patterns were analyzed for the mutant, parent strainand complemented strain by using a catalase activity staining procedure.As shown in FIG. 2A, both the parent strain A348 and the complementedstrain AG6 (pXQ9) had three distinct catalase activity bands (I, II andIII), whereas the mutant AG6 had only one band (I). This demonstratedthat the transposon insertion at the katA gene in AG6 knocked out twocatalase activity bands.

To investigate whether these two catalase activity bands originated fromthe same katA gene, different amounts of the bacterial cell extractswere loaded on the polyacrylamide gels for the catalase activitystaining. It was found that the catalase activity band III disappearedeven in A348 when less amount of the cell extract was loaded (FIG. 2B).This suggests that the catalase activity bands II and III originatedfrom the same katA gene product. The catalase activity band III appearedonly when a sufficient amount of cell extract was loaded, suggestingthat the band III was an aggregated form of catalase activity band II.

It was important to determine whether this katA gene encoded a proteinthat possessed both catalase and peroxidase activities like some of thecatalase genes (Loewen, 1997). When the peroxidase activities with thesame cell extracts were stained (Gregory and Fridovich, 1974), noperoxidase activity was found to be associated with the catalaseactivity bands. Taken together, these suggest that the katA gene encodesa catalase isozyme in the A. tumefaciens cells.

EXAMPLE 3 Determination of katA-gfp Expression Based on GFP

To study the katA gene expression in different growth conditions, A.tumefaciens strains A348 and AG6 were grown at 28° C. for 24 hr on agarplates of MG/L, AB, IB (Cangelosi et al., 1991), and fresh Kalanchoeleaf tissue and stem tissue sections which were sterile and placed on MSmedium (Murashige and Skoog, 1962). The cells were harvested, washed anddiluted to a concentration of approximately OD₆₀₀=0.5. The fluorescenceof each cell suspension was measured by Luminescence Spectrometer LS50B(Perkin Elmer) using A348 as the blank. The excitation wavelength was423 nm; the emission wavelength was 509 nm. The fluorescence levels wereexpressed as the fluorescence values divided by the corresponding OD₆₀₀.

To study the katA gene expression in different genetic backgrounds, A.tumefaciens strains A348, AG6, AG6 (pSW172), AG6 (pXQ9), A613, CGI1 andCGI1 (pXQ9) were grown at 28° C. for 24 hours on IB (pH 5.5) agarplates. The fluorescence of each strain was determined as describedabove.

To determine if H₂O₂ can induce the katA expression, AG6 was grown inMG/L liquid medium overnight at 28° C. The cells were harvested andresuspended in fresh MG/L liquid medium to a final concentration ofOD₆₀₀=0.5. Aliquots (2 ml) of the cultures were transferred into steriletubes, and H₂O₂ was added to the tubes to final concentrations of 30 μM,60 μM and 120 μM. The cell suspensions were incubated at 28° C. for 2hours. Then 1 ml of each cell suspension was centrifuged, washed andresuspended in the Laemmli (1970) sample buffer, and subjected toWestern blot as described later.

EXAMPLE 4 Mutagenesis

The C-terminus of KatA was deleted by PCR to generate pXQ11 and pXQ22.Site-directed mutagenesis of katA was performed by overlap extension PCR(Ho et al, 1989). Four oligonucleotides were designed to mutate a singleone amino acid of the KatA protein. Two residues Arg 94 and His 98 inthe putative catalase motif are highly conserved. His 98 was changed toAsp; alternatively, Arg 94 was changed to Gln and His 98 was changed toAsp. A 450 bp NsiI -AatII PCR fragment containing a single mutation atHis 98 or double mutations at both His 98 and Arg 94 was used to replacethe corresponding NsiI -AatII fragment of pXQ13 containing the wild-typekatA, in order to generate pXQ24 or pXQ25, respectively. The presence ofthe expected point mutations in NsiI-AatII fragment was confirmed by DNAsequencing using the ABI PRISM 377 DNA Sequencer. Similarly, A 670 bpMfeI-AatII PCR fragment containing an introduced stop codon at Ser 5 ora frameshift deletion at the second codon of the katA ORF was used toreplace the corresponding MfeI-AatII fragment of pXQ13 containing thewild-type katA, to generate pXQ28 and pXQ29, respectively.

EXAMPLE 5 Purification of His-KatA Fusion Protein and Generation ofAntibody to KatA

To generate a (His)₆-KatA fusion construct, an oligomer (containing XhoIsite) complementary to the start of the KatA ORF and an oligomer(containing KphI site) was used to amplify the KatA ORF fragment. The2.17 kb XhoI-KpnI fragment was inserted in-frame downstream of (His)₆harbored on pRSETA. The resulting pXQ15 was introduced into BL21 bytranformation. BL21 (pXQ15) was grown overnight in LB medium in thepresence of 100 μg/ml of carbenicillin at 37° C. The cell culture washarvested. Purification of (His)₆-KatA was conducted with TALON metalaffinity resin according to the manufacturer (Clontech). The purifiedprotein was injected into rabbits to generate the primary antibody.Protein analysis of KatA or GFP was carried out as described above.

EXAMPLE 6 Complementation

Plasmids pXQ13, pXQ24, pXQ25, pXQ28 and pXQ29 were digested by ClaI andligated with ClaI digested pSW172 to generate pXQ23, pXQ26, pXQ27 pXQ30and pXQ31. Plasmids pSW172, pXQ11, pXQ22, pXQ23, pXQ26, pXQ27, pXQ30 andpXQ31 were introduced into the mini-Tn5 mutant AG6 by triparental mating(Ditta et al., 1980) or electroporation (Cangelosi et al., 1991).Plasmid pXQ13 was introduced into AG6 by electroporation, followed byselection in the presence of carbenicillin. The resulting strain AG613was obtained that underwent a single crossover homologous recombinationat the katA locus; it was confirmed by Southern analysis. Thesetransformant strains were analyzed for the GFP and KatA expressionlevels.

The intracellular concentrations of H₂O₂ were measured according to thetechnique described above in Example 1 (“Measurement of intracellularH₂O₂concentrations”).

The catalase activity in whole bacterial cells was determined asdescribed above in Example 1 (“Catalase activity assay”).

EXAMPLE 7 Intercellular Repression of katA-gfp Expression

To determine whether catalase activity of one bacterial cell couldaffect the katA-gfp gene expression of another cell, AG6 was co-culturedwith other bacteria which contained catalase activity. The bacterialcells grown overnight were suspended in IB liquid and adjusted toOD₆₀₀=1.0. The AG6 cell suspension was mixed with another bacterialsuspension at 1:1 ratio. An aliquot of 12 μl bacterial suspension of asingle strain or a mixture of two strains was spotted onto IB plates.The plates were incubated overnight at 28° C.

The bacterial fluorescence under UV light was photographed and theintensity for GFP expression was measured by the procedure describedearlier. To check the growth viability of each strain in the co-culturemixture, a portion of each co-culture mixture was harvested to test theviable cell count on MG/L (for total viable cell count) and MG/Lsupplemented with 100 μg/ml kanamycin (for AG6 viable cell count)

EXAMPLE 8 katA is Inducible by Acidic pH

A. tumefaciens mutant AG6 contained a mini-Tn5 transposon containing apromoter-less green fluorescent protein (GFP) variant; the mini-Tn5transposon was inserted at 995 bp downstream from the start codon ofkatA. Since the gfp gene was under the control of the katA promoter(designated as katA-gfp), the katA expression would lead to theaccumulation of GFP, which could be visualized as bright greenfluorescence under UV light. Therefore, the differential expression ofkatA in A. tumefaciens could be determined by measuring the GFPexpression of the mutant AG6 in different conditions.

The katA-gfp expression was examined by growing AG6 on different growthmedia, MG/L (a rich medium; pH 7.0), AB (a minimal medium; pH 7.0), andIB (a minimal medium; pH 5.5), as well as on fresh Kalanchoe leaf tissueand stem tissue sections. As shown in FIG. 3, the fluorescence level ofthe bacteria grown on IB was about 10-20 fold higher than that onneutral pH media including AB and MG/L. The fluorescence levels onKalanchoe leaf tissue and stem tissue sections were about 5-10 foldhigher than those in the neutral pH media. This indicates that katAmight be induced by acidic pH, as the plant tissues also have acidic pHand minimal nutrition (Li et al., 1999).

Previous experiments have demonstrated that IB medium is representativeof the growth conditions the bacteria encounter inside plant tissuesduring the infection process (Li et al., 1999). To confirm that acidicpH can induce the katA expression, the fluorescence level on IB (pH 5.5)was compared with that on the medium having the same IB ingredients butwith the pH adjusted to pH 7.0 (IB pH 7.0). As shown in FIG. 3, thefluorescence level on IB (pH 7.0) was reduced to a level that wassimilar to other neutral media, including AB and MG/L. This demonstratesthat acidic pH can induce the katA expression.

EXAMPLE 9 Repression of katA-gfp Expression by katA

As shown earlier, the plasmid pXQ9 which carried a full-length katA genecould fully complement the katA mutation. When the fluorescence level inthe complemented strain AG6 (pXQ9) was analyzed, it was surprisinglyfound that this strain had a highly reduced fluorescence (60-70 foldreduction, as shown in FIG. 4). It appeared that the wild type katAcould repress the katA-gfp expression.

To determine if katA could specifically repress katA-gfp, pXQ9 wasintroduced into a different mini-Tn5 transposon mutant strain CGI1,which contained the transposon insertion at a chromosomal gene aopB andcould produce bright green fluorescence under UV light. As shown in FIG.4, the katA gene did not repress the aopB-gfp expression. This suggeststhat katA can specifically repress katA-gfp expression.

It was of interest to determine whether the copy number and the locationof katA could affect the ability to repress. katA was integrated intothe chromosome of AG6 through single-crossover homologous recombination.The resulting strain AG613 contained a single copy of the wild-type katAand a single copy of the katA-gfp fusion as verified by Southernanalysis. AG613 had a very low level of katA-gfp expression, just likeAG6 (pXQ9) harboring the katA gene on a plasmid (FIG. 4). This suggeststhat only one copy of katA was sufficient to repress the katA-gfpexpression, no matter whether katA is located on plasmid or chromosome.

EXAMPLE 10 Requirements of the katA-gfp Repression

It was of interest to determine if the repression by katA occurred atthe mRNA level or protein level. Site-directed mutagenesis was conductedto generate mutants that produced no or truncated KatA protein. The C atthe nucleotide position 14 within the katA open reading frame (ORF) waschanged to G. This created a stop codon at Ser 5 [designated as ΔkatA(*5)]; the resulting plasmid was named (pXQ31) (FIG. 5; Table 1). Aframeshift deletion at katA was then created by deleting the G of thesecond codon in the katA ORF [designated as ΔkatA (˜2)]; the resultingplasmid was named pXQ30 (FIG. 5).

As shown in FIG. 6, the pXQ31 construct did not generate any KatAprotein. pXQ30 generated a trace amount of KatA-like protein, presumablyproduced from an alternative translation site downstream from the startcodon or due to infrequent frame shifting of the ribosome, which couldrestore the translation of the protein. These two constructs did notrepress the katA-gfp expression as determined by both the GFPfluorescence and western analysis using the antibody to GFP (FIG. 7).This suggests that production of the KatA protein is required for therepression.

It was then important to determine whether a full-length KatApolypeptide was required for the repression. The 86 amino acids(designated as KatAΔ86) and 50 amino acids (KatAΔ50) at the C-terminusof KatA were deleted to generate pXQ11 and pXQ22, respectively (FIG. 5).These constructs produced smaller sizes of KatA proteins as expected(FIG. 6B, lanes 3 and 4). However, the amounts of these truncated KatAproteins were much less than the wild type KatA (harbored on pXQ9) (lane2), indicating that these truncated KatA proteins were unstable. Thesetruncated proteins did not repress the katA-gfp expression at asignificant level (FIG. 7, lanes 9 and 10), presumably because they didnot exhibit any significant catalase activity (FIG. 8; Table 2).

TABLE 2 Catalase activity in whole bacterial cells containing the wildtype or mutant katA genes^(a) Catalase activity Strain Protein expressed(unit/10⁸ cells) AG6(pXQ23) KatA 77145.5 ± 440.3  AG6(pXQ9) KatA 3997.3± 459.1 A348 KatA 1486.2 ± 149.5 AG6(pXQ26) KatA(98H/D)  219.3 ±19.5^(b) AG6(pXQ11) KatAΔ86   193.6 ± 25.2^(c ) AG6(pXQ27)KatA(94R/Q)(98H/D)   184.0 ± 25.1^(c ) AG6(pXQ22) KatAΔ50   182.7 ±21.5^(c ) AG6 KatA   175.2 ± 20.2^(c ) ^(a) Agrobacterium tumefacienscells were grown on IB plates and then harvested. The catalaseactivities in whole bacterial cells were measured as described in theMaterials and Methods. ^(b)The catalase activity of AG6(pXQ26) wassignificantly different from that of AG6(P < 0.05) based on Student'st-test. ^(c)The catalase activity of AG6(pXQ11), AG6/(pXQ27) orAG6/(pXQ22) was not significantly different from that of AG6 based onStudent's t-test.

Since the truncated KatA proteins did not possess any catalase activitydetected by an isozyme staining procedure (FIG. 8), it was important toknow whether a functional catalase activity was required for thisfeedback repression. The amino acid sequence of A. tumefaciens catalaseKatA was analyzed by a motif search program(http://www.motif.genome.ad.jp). It revealed that a motif of 12 aminoacids (VGMMARVTWHAA) located from amino acid 89 to 100 from the startcodon was qualified for a peroxidases active site signature. This motifmight be involved in the catalase activity.

Site-directed mutagenesis was conducted to inactivate the catalaseactivity. Computer analysis revealed that Arg 94 and His 98 in theconservative motif of KatA might be crucial for the catalase activity.Previous studies have indicated that the corresponding residues Arg 102and His 106 of the E. coli homolog HPI are important for the catalaseactivity (Hillar et al., 2000). His 98 was changed to Asp [designated asKatA (98H/D)]; Arg 94 was changed to Gln and His 98 was changed to Asp[designated as KatA (94R/Q) (98H/D)]. The resulting plasmids were namedpXQ26 and pXQ27, respectively.

As shown in FIG. 8, both the wild-type strain A348 and the complementedstrain AG6 (pXQ23) had the KatA catalase activity bands, whereas theywere missing in the mutant AG6, AG6 (pXQ26) and AG6 (pXQ27). Thissuggests that alteration of His 98 or both Arg 94 and His 98 in theconservative motif of KatA abolished the KatA catalase activity detectedby the staining procedure. Western blot analysis was performed to checkthe stability of the mutant proteins. As shown in FIG. 6A, AG6 (pXQ26)and AG6 (pXQ27) produced the same size of KatA proteins as the wildtype, and a high level of the point mutant proteins accumulated in thecells. As shown in FIG. 7, these two point mutant proteins couldslightly repress the katA-gfp expression. The GFP expression wasvirtually undetectable in AG6 (pXQ23) (lane 4), but it was reduced inAG6 (pXQ26) and AG6 (pXQ27) (lanes 5 and 6), as compared with thestrains expressing no or truncated protein KatA (lanes 7, 8, 9 and 10).

It was important to determine if the mutant KatA proteins possessed anytrace amount of catalase activity. The genes encoding those mutantproteins were introduced into AG6 that lacks katA. Then the catalaseactivity in whole bacterial cells was measured, because this couldpresumably avoid any inactivation of catalase activity due to the cellbreak-up process.

As shown in Table 2, the catalase activity in the bacteria containingthe mutant protein, KatAΔ86, KatA (94R/Q) (98H/D) or KatAΔ50 wasslightly higher than that in AG6, but not at a statistically significantlevel. This suggests that these mutant KatA proteins did not possess anysignificant catalase activity. The activities in whole cells for thosebacteria were apparently due to the catalase other than KatA, since AG6(which lacks katA) possessed catalase activity (FIG. 8 and Table 2). Thecatalase activity in AG6 (pXQ26) was statistically higher than that ofAG6, suggesting that KatA (98H/D) possessed a low level of catalaseactivity. This low activity presumably has contributed to the lowrepression of katA-gfp (FIG. 7).

It is interesting to note that KatA (94R/Q) (98H/D) [in the strain AG6(pXQ27)] repressed the expression of katA-gfp at a low level (FIG. 7),since the activity for this protein was not statistically significantand was lower (if any) than that of KatAΔ86 (Table 2), which did notrepress the katA-gfp expression at a significant level (FIG. 7). Thissuggests that both the KatA catalase activity and the protein itselfwere involved in the repression of katA-gfp.

In summary, the repression of katA-gfp expression required theproduction of a functional KatA protein that possessed the catalaseactivity. In addition, among the mutant KatA proteins only KatA (98H/D)and KatA (94R/Q) (98H/D) could significantly repress the katA-gfpexpression at a level much lower than the wild-type KatA (FIG. 7).Incidentally, only these two mutant KatA proteins accumulated at a veryhigh level in the bacteria (FIG. 6). This suggests that the sheer amountof the KatA proteins present in the cells might contribute to therepression, presumably because the mutant KatA proteins could still bindto H₂O₂ to reduce the availability of intracellular H₂O₂ to inducekatA-gfp expression. KatA (98H/D) repressed the katA-gfp expression at alevel slightly higher than KatA (94R/Q) (98H/D) (FIG. 7), presumably dueto its low catalase activity while KatA (94R/Q) (98H/D) did not possessany significant catalase activity (Table 2) because both two importantresidues at the putative active site have been altered (FIG. 5).

EXAMPLE 11 katA Can Be Induced by Hydrogen Peroxide

It was then important to determine how the catalase activity repressedthe katA-gfp expression. One possibility is that catalase may reduce theintracellular H₂O₂ levels to repress the katA-gfp expression. Todetermine whether the expression of katA can be induced by H₂O₂, likesome of other bacterial catalases (Loewen, 1997), AG6 was treated withlow concentrations of H₂O₂ (30-120 μM) in liquid MG/L medium and thekatA-gfp expression levels were then determined.

As shown in FIG. 9, the katA-gfp expression levels increasedsignificantly in the presence of H₂O₂, suggesting that the katAexpression could be induced by H₂O₂. This indicates that the reason thatthe catalase activity could repress the katA-gfp expression is thatcatalase could deplete the katA inducer level. This implies that theendogenous H₂O₂ acts as the intracellular inducer for the katAexpression inside A. tumefaciens cells and that induction of katA byacidic pH involves the increase of intracellular H₂O₂ levels.

EXAMPLE 12 Acidic pH Could Enhance the Accumulation of IntracellularH₂O₂ in katA Cells

Since katA could be induced by H₂O₂, it was important to determinewhether the induction of katA by acidic pH was indeed due to theenhanced levels of endogenous H₂O₂ in the bacterial cells. Theintracellular H₂O₂ concentrations were measured for the bacteria grownon the acidic IB plates (pH 5.5) or AB plates of neutral pH (pH 7.0),according to the previously described procedures (González-Flecha andDemple, 1995; 1997) that were based on a complete equilibration of theintracellular and extracellular H₂O₂ levels of the bacterial cells thatwere grown on IB or AB and then resuspended in a phosphate buffer (pH7.4) (see the Materials and Methods).

As shown in Table 3, the intracellular H₂O₂ concentration for the AG6cells grown on IB was about 10 times higher than those for the bacterialcells containing the wild-type katA [A348 and AG6 (pXQ9)]. The level forthe AG6 cells grown on AB was about 4 times higher than those for thebacteria having the wild type katA. The level of intracellular H₂O₂increased about 3 times when the AG6 cells were switched from pH 7.0 topH 5.5. The intracellular H₂O₂ concentrations were virtually constantfor the bacteria having the wild type katA, no mater whether they weregrown on AB or IB plates. These indicate that mutation at katA enhancedthe accumulation of intracellular H₂O₂ for the bacteria grown at acidicpH. The elevated level of intracellular H₂O₂ caused by acidic pH in theabsence of functional katA induced the expression of katA-gfp.

TABLE 3 Intracellular H₂O₂ concentrations of bacteria grown on IB and ABplates^(a) H₂O₂(μM) Strain AB (pH 7.0) IB (pH 5.5) A348 0.12 ± 0.03 0.13± 0.02 AG6 0.41 ± 0.08 1.16 ± 0.14 AG6(pXQ9) 0.10 ± 0.02 0.09 ± 0.01^(a) Agrobacterium tumefaciens cells were grown on AB or IB plates. Theintracellular H₂O₂ concentrations were measured as described in theMaterials and Methods, based on a complete equilibration of theintracellular and extracellular H₂O₂ levels of the bacterial cells thatwere grown on IB or AB and then resuspended in a phosphate buffer (pH7.4).

EXAMPLE 13 Intracellular H₂O₂ Scavenging Assay Technology

The studies indicate that mutation at katA can enhance the accumulationof intracellular H₂O₂ in the bacterial cells grown at acidic pH. Theelevated level of intracellular H₂O₂ caused by acidic pH in the absenceof functional katA can induce the expression of katA-gfp. When a katAgene encoding a functional catalase is introduced into the cells lackingthe katA gene, katA-gfp expression is dramatically repressed (FIGS. 4,7, 8 and 10).

Two mutant KatA proteins truncated at the C-terminus exhibited a verylow level of accumulation in the cells and no significant catalaseactivity; neither mutant repressed katA-gfp expression at anysignificant level (FIGS. 7 and 8; Table 2). These indicate that thekatA-gfp expression levels can reflect the intracellular H₂O₂ scavengingcapacity of the cells. If the cells are introduced with molecules, largeor small, that can scavenge the intracellular H₂O₂ levels, the katA-gfpexpression will be repressed. Therefore, measuring the katA-gfpexpression can monitor the intracellular H₂O₂ scavenging capacity of amolecule that is introduced into the cells.

In the present study, the fully functional catalase gene katA repressedthe katA-gfp expression at the highest level; thus it has the highestcapacity to scavenge intracellular H₂O₂ (FIGS. 4, 7 and 10). Two mutantKatA proteins truncated at the C-terminus exhibited a very low level ofaccumulation in the cells and no significant catalase activity; neitherrepressed the katA-gfp expression at any significant level (FIGS. 7 and8; Table 2). Thus, they have no significant capacity to scavengeintracellular H₂O₂. Two other mutant KatA proteins, KatA (98H/D) andKatA (94R/Q) (98H/D), could significantly repress the katA-gfpexpression at a level much lower than the wild-type KatA (FIG. 7).

Incidentally, only these two mutant KatA proteins accumulated at a veryhigh level in the bacteria (FIG. 6). This suggests that the sheer amountof the KatA proteins present in the cells might contribute to therepression, presumably because the mutant KatA proteins could still bindto H₂O₂ to reduce the availability of intracellular H₂O₂. KatA (98H/D)repressed the katA-gfp expression at a level slightly higher than KatA(94R/Q) (98H/D) (FIG. 7), presumably due to its low catalase activitywhile KatA (94R/Q) (98H/D) did not possess any significant catalaseactivity (Table 2). All of these indicate that this assay technology isextremely sensitive in determining the intracellular H₂O₂ scavengingcapacities of different molecules and that this assay can analyzemolecules that can scavenge intracellular H₂O₂ directly or indirectly.

EXAMPLE 14 Repression of katA-gfp Expression by Surrounding BacterialCells

To investigate whether the repression of katA-gfp expression could occurintercellularly, AG6 was co-cultured with the wild type stain A348 (seeExample 7). As shown in FIG. 10, A348 indeed could repress the katA-gfpexpression in the surrounding AG6 cells, when A348 was mixed with AG6(upper panel). The fluorescence level of the A348+AG6 mixture wasreduced about 20 fold as compared to that of AG6 alone (lower panel).Evaluation of the viable cell count of the AG6+A348 mixture showed thatAG6 was 50% of the total cells as expected, suggesting that therepression in the mixture was caused intercellularly and not simply dueto the dilution effect from the non-fluorescent A348 cells.

This intercellular repression phenomenon was also observed when AG6 wasco-cultured with R. meliloti and E. coli which contained the catalasegenes (Herouart et al, 1996; Loewen, 1997) and possessed the catalaseactivity as measured by the catalase activity assay. The repression ofkatA-gfp expression by R. meliloti and E. coli was weaker than that ofA348 (FIG. 10), presumably because they grew slower than AG6 on IB plateas demonstrated by the viable cell count experiment. These suggest thatH₂O₂ generated in AG6 could pass through the bacterial cell membranesand enter into the surrounding cells that possessed the catalaseactivity equivalent to KatA. The repression of katA-gfp expression couldbe achieved by catalases from other bacterial species. This indicatesthat intracellular H₂O₂ scavengers can be applied, external and adjacentto the cells under oxidative stress, in the form of live cells ornon-living systems that can receive H₂O₂ from the adjacent cells underoxidative stress.

Interestingly, active catalase added to the medium of AG6 cells did notrepress katA-gfp expression. Catalase purchased from Sigma was addedinto IB plates at a final concentration of 1 or 5 microgram per ml. AG6cells (carrying the katA-gfp fusion) were then grown on the IB plate.The bacterial cells exhibited the same level of GFP expression as theAG6 cells grown in the absence of catalase. To ensure that the catalasein the IB medium was still active, hydrogen peroxide was dropped ontothe catalase-containing plate. Air bubbles were instantly visible in thecatalase-containing plate; no air bubble was found in the controlcatalase-free IB plate. This demonstrated that the catalase in themedium was active. Therefore, it can be concluded that extracellularcatalase could not remove the intracellular hydrogen peroxide.

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While a number of embodiments have been presented, it is apparent thatthe basic construction can be altered to provide other embodiments whichutilize the methods and kits of the invention. The scope of theinvention is to be defined by the claims appended hereto rather than thespecific embodiments presented herein by way of example.

1. A method for determining the ability of a compound to remove areactive oxygen species (ROS), comprising: a) providing a cell ofAgrobacterium tumefaciens that is genetically modified to provide aconstitutively elevated level of said ROS and comprising a promoter of aKatA gene that is inducible by H2O2 and is operably linked to a reportergene, wherein the reporter gene is heterologous to the KatA genepromoter, b) exposing the cell to a compound potentially able to removethe ROS; c) measuring change in ROS-inducible expression of the reportergene in the cell when exposed to the compound, wherein the KatA promoteris induced by exposing the cell to acidic culture conditions before step(c).
 2. A method for selecting a nucleic acid encoding a protein whichis able to remove a reactive oxygen species (ROS), from a plurality ofnucleic acids, the method comprising: a) providing Agrobacteriumtumefaciens cells that are genetically modified to provide aconstitutively elevated level of said ROS and comprising a promoter of aKatA gene that is inducible by H2O2 and is operably linked to a reportergene, wherein the reporter gene is heterologous to the KatA genepromoter; b) introducing into the cells expression vectors comprising aplurality of nucleic acids encoding proteins which are potentially ableto remove the ROS; c) measuring change in the ROS-inducible expressionof the reporter gene in the cells when nucleic acids are expressed; d)selecting for cells with reduced ROS-inducible expression of thereporter gene; and e) isolating nucleic acids from the cells withreduced expression of the reporter gene; wherein the isolated nucleicacids encodes a protein able to remove the ROS; wherein the KatApromoter is induced by exposing the cell to acidic culture conditionsbefore step (c).
 3. The method of claim 1, wherein the step of exposingthe cell to the compound comprises providing the compound externally tothe cell.
 4. The method of claim 1, wherein the step of exposing thecell to the compound comprises expressing the compound from a nucleicacid inside the cell.
 5. The method of claim 1, wherein the cell lacks agene encoding an active enzyme selected from the group consisting of:catalase, superoxide dismutase, alkyl hydroperoxidase, and glutathionereductase.
 6. The method of claim 5 wherein the active enzyme iscatalase.
 7. A diagnostic kit for determining the ability of a geneproduct to remove a reactive oxygen species (ROS), the kit comprising:a) an Agrobacterium tumefaciens cell that is genetically modified toprovide a constitutively elevated level of said ROS comprising apromoter of a KatA gene that is inducible by H2O2 and is operably linkedto a reporter gene, wherein the reporter gene is heterologous to theKatA gene promoter, b) means for introducing into the cell a nucleicacid encoding a gene product potentially able to remove the ROS; and c)instruction for determining a reduction in expression of the reportergene in cell of (a) when the nucleic acid is expressed, therebydetermining whether the gene product is able to remove the ROS, saidinstruction further includes instructing a step of inducing the KatApromoter by exposing the cell to acidic conditions, before step (c). 8.The diagnostic kit of claim 7, wherein the reporter gene encodes areporter product, and wherein the kit further comprises means formeasuring the reporter product.
 9. The kit of claim 7, furthercomprising means for elevating the intracellular level of the ROS in thecell.
 10. The kit of claim 7, wherein the cell lacks a gene encoding anactive enzyme selected from the group consisting of: catalase,superoxide dismutase, alkyl hydroperoxidase, and glutathione reductase.11. The kit of claim 10, wherein the active enzyme is catalase.
 12. Themethod of claim 2, wherein the cell lacks a gene encoding an activeenzyme selected from the group consisting of: catalase, superoxidedismutase, alkyl hydroperoxidase, and glutathione reductase.
 13. Themethod of claim 12, wherein the active enzyme is catalase.
 14. Themethod claim 2, wherein the reporter gene encodes a protein functionalin the bacterial cell.